Abstract The P450 system includes a variety of enzymes involved in the elimination of a host of foreign compounds from the body. The purpose of the P450 system is to catalyze oxygenation reactions, commonly generating products that are more water-soluble and readily excreted. In some cases, however, the products are reactive ? capable of binding to DNA, RNA or critical proteins ? and leading to mutagenesis, carcinogenesis and other toxicities. The P450s involved in xenobiotic metabolism are terminal components of an electron transfer chain found in the endoplasmic reticulum (ER) ? P450 does not function alone but must receive two electrons from their redox partners NADPH-cytochrome P450 reductase (CPR) and/or cytochrome b5. Although cytochrome b5 can transfer the second electron efficiently, CPR is generally acknowledged as the primary electron source and can support the monooxygenase reaction alone. When present in microsomes, P450 is in a large excess over CPR. Although this ratio varies depending on induction status, P450 levels exceed those of CPR by a 7:1 to 20:1 ratio. Since CPR and P450 form a 1:1 functional complex, CPR must be capable of supplying electrons to many different P450s. In the event that particular P450s have a higher affinity for association with CPR, then electrons would preferentially flow to those P450s. Consequently, P450s less able to compete for limiting CPR must have mechanisms that allow them to receive electrons; otherwise they would be metabolically silent. These conditions raise questions regarding how the components of the P450 system are organized in the membrane. P450 system proteins are crowded in the ER, providing many opportunities to interact. It is now established that P450 enzymes form both homomeric and heteromeric complexes, and that these complexes affect P450 function. As an additional complication, the P450 system proteins reside in a membrane that is heterogeneous in nature, where the characteristics of the membranes can affect protein organization. Consequently, the P450 system needs to be considered not simply as a series of monomeric proteins interacting with their redox partners, but as components of a supramolecular complex that is affected by its membrane environment. The objective of this proposal is to better understand how the proteins of the P450 monooxygenase system are organized in the ER and the role of P450-P450 interactions on the function of these enzymes. This will be accomplished by addressing the following Specific Aims: Aim 1 ? to characterize the physical complexes among CPR and multiple P450s and to identify the protein regions responsible for P450P450 complex formation; and Aim 2 ? to identify where P450 system proteins reside in the ER, how ER heterogeneity influences P450 system protein localization, and what protein regions are responsible for localization in specific membrane domains. These studies will increase our understanding of how the P450 system proteins interact in biological membranes, and how the membrane can affect these responses ? which will have a significant impact on generation of toxic metabolites from xenobiotics.